N-terminus plus linker domain of Mg-chelatase D subunit is essential for Mg-chelatase activity in Oryza sativa.
Biochem Biophys Res Commun
; 497(2): 749-755, 2018 03 04.
Article
em En
| MEDLINE
| ID: mdl-29462612
ABSTRACT
Mg chelatase, a key enzyme in chlorophyll biosynthesis, is comprised of I, D and H subunits. Among these subunits, the D subunit was regarded to mediate protein interactions due to its unique protein domains. However, the functional roles of the different domains of the D subunit in vivo remain unclear. In this study, we dissected the rice (Oryza sativa) D subunit (OsCHLD) into three peptide fragments the putative chloroplast transit peptide (TP, Met1 to Arg45), the N-terminus plus linker domain (OsCHLDN + L, Ala46 to Leu485) and the C-terminus (OsCHLDC, Ile486 to Ser754), to explore the roles of these fragments. The results of the yeast two-hybrid assay and the in vitro reconstitution of the Mg-chelatase activity showed that only OsCHLDN + L interacted with the I and H subunits and maintained most of the Mg-chelatase activity in vitro. Furthermore, artificial TP-OsCHLDNâ¯+â¯L and TP-OsCHLDC were overexpressed in rice. Interestingly, an incomplete co-suppression had occurred in both of the overexpressed (OsCHLDNâ¯+â¯L-ox and OsCHLDC-ox) plants, resulting in a significantly downregulated expression of endogenous OsCHLD. Therefore, these transgenic plants had adequate OsCHLDN + L and OsCHLDC instead of endogenous OsCHLD, providing ideal models to study the function of different domains of the D subunit in vivo. The OsCHLDNâ¯+â¯L-ox plants showed an identical phenotype to that of the wild type, while the OsCHLDC-ox plants demonstrated a yellowish phenotype that resembled the D subunit mutants. These results indicated that only OsCHLDN + L could complement the function of endogenous OsCHLD, providing direct evidence that OsCHLDN + L is essential for Mg-chelatase activity in vivo.
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MEDLINE
Assunto principal:
Proteínas de Plantas
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Oryza
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En
Revista:
Biochem Biophys Res Commun
Ano de publicação:
2018
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Article